Targeted skipping of a single exon harboring a premature termination codon mutation: Implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients
Targeted skipping of a single exon harboring a premature termination codon mutation: Implications and potential for gene correction therapy for selective dystrophic epidermolysis bullosa patients
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DOI：
10.1038/sj.jid.5700435
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发表时间：
2006-12-01
影响因子：
6.5
通讯作者：
Shimizu, Hiroshi
中科院分区：
文献类型：
--
作者：
Goto, Maki;Sawamura, Daisuke;Shimizu, Hiroshi
本研究旨在探讨反义寡核苷酸（AON）治疗营养不良性大疱性表皮病（DEB）的可行性。AON被设计为诱导含有提前终止密码子突变的靶向外显子的跳跃，导致开放阅读框的恢复。我们靶向COL7A1的外显子70，因为日本DEB患者中的复发突变5818delC定位于外显子70。我们发现，一个AON诱导有效的跳跃正常外显子70含有16个氨基酸。附着和迁移分析表明，重组胶原蛋白没有外显子70的贡献是类似的效果正常的VII型胶原蛋白。接下来，我们通过在5818处缺失胞嘧啶来合成突变特异性AON。将该AON引入到携带5818delC的DEB角质形成细胞中表明，AON诱导异常5818delC等位基因中的外显子70的跳跃。此外，6.2%的DEB角质形成细胞在应用突变特异性AON后开始在体外表达VII型胶原。将AON注射到移植有DEB角质形成细胞和成纤维细胞的大鼠模型中诱导少量的VII型胶原蛋白表达。我们的结论是使用突变特异性AON跳过靶向外显子可能显示出未来DEB患者基因治疗的潜力。
This study examined the feasibility of antisense oligoribonucleotide (AON) therapy for dystrophic epidermolysis bullosa (DEB). AON was designed to induce skipping of a targeted exon containing a premature termination codon mutation, resulting in restoration of the open reading frame. We targeted exon 70 of COL7A1, as a recurrent mutation 5818delC in Japanese DEB patients was localized to exon 70. We found that one AON induced effective skipping of normal exon 70 containing 16 amino acids. Attachment and migration analyses showed that recombinant collagen without contribution of exon 70 was similar in effect to normal type VII collagen. Next, we synthesized mutation-specific AON by deleting cytosine at 5818. Introduction of this AON into DEB keratinocytes harboring 5818delC showed that the AON induced skipping of exon 70 in the abnormal 5818delC allele. Furthermore, 6.2% of DEB keratinocytes started to express type VII collagen in vitro after application of the mutation-specific AON. Injection of the AON into rat model grafted with DEB keratinocytes and fibroblasts induced a low amount of type VII collagen expression. We conclude that skipping of targeted exons using mutation-specific AON may show potential for future gene therapy for DEB patients.